Core Protein Precursor Research Articles

The multistep proteolytic maturation pathway utilized by vaccinia virus P4a protein: A degenerate conserved cleavage motif within core proteins

The most abundant vaccinia virus (VV) core protein found within the virion is protein 4a, which represents approximately 14% of the particle's dry weight. The 4a protein is synthesized as a 102.5-kDa precursor, which is proteolytically processed to a 62-kDa product concomitant with virion assembly. To identify the pathway by which P4a is converted into 4a, immunological reagents which are specific for subregions of the P4a precursor were developed and used in concert with peptide mapping and protein sequencing procedures. The result obtained suggest that the 891 amino acid P4a precursor is cleaved at two locations, between residues 614 and 615 and 697 and 698. Both the large amino-terminal 4a protein (residues 1–614) and the carboxy-terminal-derived 23-kDa protein (residues 698–891) become major virion contituents. The location and fate of the small internal peptide (residues 615–697) is not known. Interestingly, an analysis of the predicted amino acid sequences at the sites of cleavage within the P4a precursor indicated the presence of an Ala-Gly ↓ Thr motif flanking the 697–698 site and an Ala-Gly ↓ Ser motif flanking the 614–615 site. Since both of these signals are quite similar to the Ala-Gly ↓ Ala signal previously identified as the cleavage point within the VV P4b and P25K core protein precursors (VanSlyke et al., 1991. J. Gen. Virol. 72, 411–416), this suggests that processing of all three core protein precursors may be coordinately linked and/or catalyzed by the same proteinase during viral assembly.

The proteinase polypeptide of adenovirus serotype 2 virions

The Ad2 proteinase, which is thought to be encoded by a 23-kDa open reading frame located at the end of the L3 family of late mRNAs, is expressed poorly even late after infection. To obtain sufficient proteinase for biochemical characterization, a DNA fragment containing the 23-kDa open reading frame was cloned into plasmids that permit efficient expression in Escherichia coli. Polyclonal antiserum specific for the Ad2 proteinase was produced by immunizing rabbits with a fusion protein that included the entire proteinase open reading frame, and this antiserum was used to show that the product of the 23-kDa reading frame is assembled into virions. Bacterial products corresponding to the complete 204 amino acid proteinase reading frame, to a 9 amino acid proteinase deletion, and to a proteinase fusion protein of 227 amino acids were used to determine the size of the proteinase polypeptide in Ad2 virions and in infected HeLa cell extracts. A single proteinase polypeptide that migrated during SDS-polyacrylamide gel electrophoresis with the 204 amino acid recombinant proteinase was detected in wild-type and H2ts1 virions, and in infected cell extracts. Immunoblot titrations showed that a wild-type Ad2 virus particle contains about 10 proteinase polypeptides; an H2ts1 virion has f̃ivefold less proteinase. In virions, the proteinase was associated primarily with the virus core. The 204 amino acid proteinase produced in E. coli permitted cleavage of the major core protein precursor, P-VII, to mature, authentic VII, but the proteinase deletion lacking 9 amino acids from near the amino-terminus was inactive. These results are inconsistent with autocatalytic processing of the Ad2 proteinase as was reported by Chatterjee and Flint (1987, Proc. Natl. Acad. Sci. USA 84, 714–718).

Identification and sequencing of the spheroidin gene of Choristoneura biennis entomopoxvirus

Entomopoxviruses are a class of insect viruses whose virions are embedded in cytoplasmic occlusion bodies. The major component of these protective complexes is a protein called spheroidin. An open reading frame encoding the spheroidin gene of Choristoneura biennis entomopoxvirus has been identified and sequenced in our laboratory. This protein coding region is 1023 nucleotides long and specifies a polypeptide of 38,500 Da. Spheroidin was purified by SDS polyacrylamide gel electrophoresis, electroeluted, and its amino terminus sequence was determined on a gas phase sequencer. We observed that the first 20 N-terminal amino acids were absent in the mature processed form of the spheroidin molecule. Examination of these 20 residues revealed their hydrophobic nature and close resemblance to the consensus signal peptide sequence which is commonly found on membrane proteins. The DNA sequence of the spheroidin gene predicted a processed polypeptide with a molecular weight of 36 kDa. However, spheroidin was observed to aggregate in complexes composed of 50-kDa monomers. Intermolecular disulfide bonds were shown to play major roles in the formation and structure of these viral occlusion bodies. The difference in molecular weight between the predicted protein and its counterpart in infected cells is likely due to post-translational modifications. Indeed, two potential asparagine-linked glycosylation sites are present on the spheroidin molecule. The 5' flanking regions of the spheroidin gene and the vaccinia major core protein precursor gene P4b were shown to share substantial homology.

Deglycosylation of chondroitin sulfate proteoglycan and derived peptides

In order to define the domain structure of proteoglycans as well as identify primary amino acid sequences specific for attachment of the various carbohydrate substituents, reliable techniques for deglycosylating proteoglycans are required. In this study, deglycosylation of cartilage chondroitin sulfate proteoglycan (CSPG) with minimal core protein cleavage was accomplished by digestion with chondroitinase ABC and keratanase, followed by treatment with anhydrous HF in pyridine. Nearly complete deglycosylation of secreted proteoglycan was verified within 45 min of HF treatment by loss of incorporated [3H]glucosamine label from the proteoglycan as a function of time of treatment, as well as by direct analysis of carbohydrate content and xylosyltransferase acceptor activity of unlabeled core protein preparations. The deglycosylated CSPG preparations were homogeneous and of high molecular weight (approximately 370,000). Comparison of the intact deglycosylated core protein preparations with newly synthesized unprocessed precursors (apparent Mr approximately 360,000) suggested that extensive proteolytic cleavage of the core protein did not occur during normal intracellular processing. Furthermore, peptide patterns generated after clostripain digestion of core protein precursor and of deglycosylated secreted proteoglycan were comparable. With the use of the clostripain digestion procedure, peptides were produced from unlabeled proteoglycan, and two predominant peptides from the most highly glycosylated regions (the chondroitin sulfate rich regions of the proteoglycan) were isolated, characterized, and deglycosylated. These peptides were found to follow similar kinetics of deglycosylation and to acquire xylose acceptor activity comparable to the intact core protein.

Xylosyl transfer to the core protein precursor of the rat chondrosarcoma proteoglycan

Rat chondrosarcoma chondrocytes were labeled with [3H]serine or [3H]mannose as a precursor. Intracellular proteoglycan core protein precursor was purified from cell lysates by immunoprecipitation with polyclonal antibodies against the hyaluronic acid-binding region, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core precursor was eluted from the gels and treated with alkaline borohydride in order to convert serine residues substituted with xylose or N-acetylgalactosamine to alanine (or with alkaline sulfite to convert them to cysteic acid). After acid hydrolysis, the proportions of labeled serine and alanine (or cysteic acid) were determined by high performance liquid chromatography, and the results were compared with those obtained for the completed proteoglycan molecules isolated from the same cultures. In the completed proteoglycans, about 55% of the serine residues were substituted with xylose or N-acetylgalactosamine, while the corresponding figure for the intracellular precursor molecules was less than 5%. These results indicate, in agreement with our previous kinetic data, that the major part of the xylosyl transfer to the chondrosarcoma proteoglycan core protein precursor must occur late in the processing sequence, i.e. after about 85% of its intracellular lifetime and no more than 7 min before the addition of the rest of the chondroitin sulfate chain. The ratio of [3H]mannose to [3H]fucose in the core precursor was about 19, while that for the complete proteoglycan was about 2. This indicates the presence of high mannose, N-linked oligosaccharides on the core protein precursor which are converted to the complex forms on the completed proteoglycan. These data provide further support that the core precursor resides mainly in the pre-Golgi compartment and that xylosylation occurs mainly in a Golgi compartment.

Role of human immunodeficiency virus type 1-specific protease in core protein maturation and viral infectivity.

It is generally believed that the gag gene product of human immunodeficiency virus type 1 (HIV-1) is processed into several core proteins by a virus-specific protease. We used deletion mutation analysis to study the role of HIV-specific protease in the processing of core proteins and its requirement for viral infectivity. Several mutant genomes with deletions in the protease gene were constructed. A mammalian cell line, COS-M6, transfected with the wild-type viral genome was shown to produce virions containing processed core proteins, while COS-M6 cells transfected with two mutated genomes could express only the core protein precursor, Pr56gag. The wild-type transfectant produced infectious virus; both transfectants expressing the mutated genomes also produced virions, and one of them still retained reverse transcriptase activity. However, the mutant viral particles were devoid of infectivity. Virions with a distinct central core and an electron-dense nucleoid budded out from the plasma membrane of COS-M6 cells transfected with the wild-type genome. In contrast, noninfectious virions that budded either into cytoplasmic vacuoles or out from the plasma membrane of COS-M6 cells transfected with mutant genomes contained ring-shaped nucleoids. These results indicate that the HIV-1 protease plays a role not only in the maturation of the core proteins but also in the assembly of the virus and thus is required for viral infectivity.

18] Biosynthesis of cartilage proteoglycan and link protein

This chapter describe three procedures which have proved useful in the isolation and characterization of newly synthesized proteoglycan and link protein. The first deals with the isolation of newly synthesized link protein and core protein precursor, as well as completed forms of the proteoglycan by taking advantage of their ability to be incorporated into aggregates recoverable from the bottom of CsCl density gradients. The second presents refinements of immunoprecipitation procedures, while the third describes the separation of two populations of newly synthesized aggregating proteoglycan by agarose gel electrophoresis. Bovine articular cartilage aggregating proteoglycans exhibit age-related differences. Electrophoresis in acrylamide-agarose composite gels has been used successfully to show that the matrix contains two populations of aggregating proteoglycans which are present in different ratios at different ages. Electrophoresis in 1% agarose gels has recently been found to offer a number of advantages over the acrylamideagarose composite gel procedure.

Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture

The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 μg/ml tunicamycin) disproportionately to that of general protein synthesis measured by [ 3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [ 35S]sulfate and [ 3H]glucosamine were used as labeling precursors, the ratio of 35S 3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP- N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t 1 2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-β-xyloside, however, lasted longer ( t 1 2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: (a) tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely (b) a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.

Post-translational events in proteoglycan synthesis: Kinetics of synthesis of chondroitin sulfate and oligosaccharides on the core protein

Chondrocytes isolated from the Swarm rat chondrosarcoma were incubated in culture with [1- 3H]glucose for 30 min to 8 h. Labeled proteoglycans were isolated, treated with borohydride under alkaline conditions, and the three complex sugar structures purified: N- and O-linked oligosaccharides and chondroitin sulfate chains. The amount of incorporated radioactivity into each component sugar was analyzed by HPLC after enzyme digestion and hydrolysis. The kinetic data for labeling of each sugar over the time course of the experiment were fit to first-order rate equations and the half times ( t 1 2 ) to linear labeling were calculated. The t 1 2 values were essentially the same, 5–8 min, for galactose in all three complex sugar structures and for chain glucuronic acid in chondroitin sulfate, while that for xylitol in chondroitin sulfate, 15.8 min, was significantly longer. Thus, oligosaccharide synthesis is concomitant with chondroitin sulfate chain synthesis; the addition of the chondroitin sulfate linkage galactose occurs at or nearly at the same time as chain elongation while the addition of linkage xylose residues to the core protein may precede chain synthesis by up to 8 min. Since the intracellular t 1 2 of the core protein precursor for these cells is 45 to 90 min, the data strongly suggest that the addition of xylose is not completed to any significant extent while the polypeptide is still nascent or shortly after its release into the rough endoplasmic reticulum. It is proposed that the addition of xylose to the core protein precursor is a late endoplasmic reticulum or early Golgi event. The analytical data were consistent with the presence of ester phosphate on about 80% of the xylose residues of the newly synthesized proteoglycan.

Identification from cDNA of the precursor form of a chondroitin sulfate proteoglycan core protein.

The yolk sac carcinoma cell line L2 secretes a chondroitin/dermatan sulfate proteoglycan that has an Mr 10,000 core protein and carries an average of 14 glycosaminoglycan chains. The amino acid sequence of the mature core protein has been determined from cloned cDNA (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1321-1325). From additional cDNA sequences described in this report we have identified the prepro core protein precursor of the yolk sac carcinoma chondroitin/dermatan sulfate proteoglycan. From the amino acid sequence of the core protein precursor can be deduced the protein processing events in the biosynthesis of the proteoglycan. The amino acid sequence shows that the 104-amino acid mature core protein is processed from a 179-amino acid prepro core protein precursor which, in addition to the mature core protein, contains a 26-amino acid signal peptide as well as a 49-amino acid propeptide. The molecular weight of the prepro core protein predicted from the cDNA sequence (Mr = 18,600) was in good agreement with the molecular weight of the in vitro translation product (Mr = 19,000) of hybrid-selected mRNA. Accordingly, we have designated the proteoglycan core protein PG19. Further analysis of the PG19 mRNA by RNA sequencing confirmed the identification of the core protein translation initiation codon by revealing stop codons in all three reading frames of the upstream mRNA sequence. Primer extension analyses demonstrated that the 5' untranslated sequence of the proteoglycan mRNA is approximately 220 nucleotides in length, which, combined with the length of cDNA clones, accounts for the entire length of the coding sequence of PG19 mRNA from L2 cells. The cDNA sequences presented here establish the complete protein sequence of PG19 and provide evidence of polypeptide processing during the biosynthesis of the proteoglycan core protein.

Control of proteoglycan biosynthesis. Further studies on the effect of serum on cultured bovine articular cartilage.

Proteoglycan synthesis in explant cultures of adult bovine articular cartilage is stimulated in a dose-dependent manner when the tissue is cultured in the presence of foetal-calf serum. The stimulation of proteoglycan synthesis is paralleled by a similar increase in DNA synthesis; however, when DNA synthesis is inhibited by hydroxyurea the stimulation of proteoglycan synthesis by serum remains essentially the same. The apparent half-life of the pool of proteoglycan core protein precursor was measured in freshly isolated tissue as well as in tissue cultured for 7 days in the presence and in the absence of foetal-calf serum; under all conditions the half-life was the same, suggesting that this value is independent of the net rate of proteoglycan synthesis. In the presence of actinomycin D, an inhibitor of RNA synthesis, there was a difference in the apparent half-life of the available pool of mRNA coding for proteoglycan core protein: 8.5 h for tissue maintained in the presence of serum and 3.8 h for tissue cultured in the absence of serum. It is suggested that proteoglycan synthesis is stimulated by serum factors at the level of DNA-dependent RNA synthesis. Concomitant with an increase in the rate of proteoglycan synthesis induced by the presence of serum in the culture medium, an increase in the concentrations of several glycosyltransferases involved in chondroitin sulphate synthesis was also observed.

Subcellular localization of the synthesis and glycosylation of chondroitin sulfate proteoglycan core protein.

In order to study the temporal and topological events involved in the processing and assembly of chondroitin sulfate proteoglycan, a fractionation scheme involving differential centrifugation and discontinuous sucrose density gradient centrifugation was developed for homogenates of chick embryo sternal chondrocytes. The precursors in these subcellular fractions were examined by a series of pulse, pulse-chase, and continuous labeling experiments. When chondrocytes were pulsed for 20 min with [35S]methionine, an immunoprecipitable core protein precursor with an approximate molecular size of 376,000 Da was localized to the rough endoplasmic reticulum fractions. Further incubation under chase conditions showed the presence of the 376,000-Da species as well as two additional polypeptides of higher molecular masses in the smooth membrane-enriched fractions within the next 2 h. This translocation did not occur in the presence of the energy transfer inhibitor carbonyl cyanide-m-chlorophenylhydrazone. The labeling pattern of the newly synthesized core protein precursor with either [3H] mannose or [3H]glucosamine showed that N-linked oligosaccharide addition was found on the earliest synthesized product in the rough endoplasmic reticulum, and the addition of this oligosaccharide was inhibited by co-incubation with tunicamycin. Furthermore, the high mannose oligosaccharide was susceptible to cleavage by endo-beta-N-acetylglucosaminidase H, while upon chase approximately 56 and 31% of the glucosamine- and mannose-labeled oligosaccharides, respectively, were processed to resistant forms, presumably in the Golgi complex. Both direct assay of glycosyl- and sulfotransferases requisite for addition of chondroitin sulfate chains and sensitivity of intracellular precursors to chondroitinase, keratanase , and endoglycosidase H suggest that only the N-linked oligosaccharides are added in the rough endoplasmic reticulum and glycosaminoglycan chain addition occurs predominantly in smooth membranes.

Effect of puromycin on cartilage proteoglycan structure and capacity to bind hyaluronic acid

Rat chondrosarcoma chondrocytes were cultured in the presence of puromycin to induce premature termination of core protein precursor. The structure and function of intracellular and extracellular proteoglycans were assessed by molecular sieve chromatography and polyacrylamide gel electrophoresis. [ 3H]Serine incorporation was maximally inhibited by 3 × 10 −4 m puromycin but unaffected by 10 −5 m puromycin. Proteoglycans synthesized in the presence of puromycin exhibited increased monomer size due to increased chondroitin sulfate chain size, typical of proteoglycans synthesized in the presence of protein synthesis inhibitors, but no loss in ability to bind to hyaluronic acid; and no loss in core protein size was observed after treatment with chondroitinase. These data suggest that chondrocytes select only completed or nearly completed core protein molecules to process into proteoglycans.

Localization of proteoglycan core protein in subcellular fractions isolated from rat chondrosarcoma chondrocytes.

Chondrocytes from the Swarm rat chondrosarcoma were pulse-labeled with [3H]serine for 30 min and chased, in the presence of cycloheximide, for times up to 300 min. The movement of newly synthesized core protein precursor of the proteoglycan through elements of the endoplasmic reticulum and Golgi complex was examined. Rough and smooth microsome fractions were obtained by centrifuging postmitochondrial supernatants from cell homogenates on discontinuous sucrose gradients. The core protein precursor was identified in subcellular fractions by (a) immunoprecipitation with an antiserum directed against the hyaluronate binding region of the core protein and the link protein and (b) its size on polyacrylamide gels. Labeled core protein precursor decreased from the microsomes with a t1/2 of 60 +/- 8 min, nearly the same as for the appearance of label in completed proteoglycan monomer (t1/2 = 58 +/- 13 min), consistent with a precursor-product relationship. After correcting for incomplete recovery of the core protein precursor in the microsomal fractions and for cross-contamination of the smooth microsomes by elements of rough endoplasmic reticulum, the redistribution of core protein precursor and completed proteoglycan in the intracellular compartments and of labeled extracellular proteoglycan were fit to a three-compartment model. A t1/2 of 98 +/- 7 min for the loss of core protein precursor from the rough microsomes and a t1/2 = 10 +/- 4 min for the completed proteoglycan in the intracellular compartment (Golgi and secretory vesicles) was obtained. The data indicate that at least 70% of the intracellular transit time for the core protein precursor is spent in the rough endoplasmic reticulum. The addition of glycosaminoglycan chains followed by secretion from the cell occurs relatively rapidly, occupying less than 30% of the total intracellular dwell time.

Cytoskeleton-associated Pr65 gag and assembly of retrovirus temperature-sensitive mutants in chronically infected cells

Certain temperature-sensitive ( ts) mutants of murine leukemia virus (MuLV) were observed to be defective in virus assembly. These mutants also accumulated intracellular core protein precursor, Pr65 gag , at 39°, the nonpermissive temperature. At 39°, virions released from cells infected with the various is mutants also contained elevated levels of Pr65 gag relative to virions released at 33°, the permissive temperature. Detergent extraction of pulse-labeled cells with Nonidet P-40 (NP-40) generated an NP-40-insoluble cytoskeleton-enriched fraction. Reextraction of this fraction with deoxycholate followed by gel electrophoresis of solubilized, immunoprecipitated viral proteins showed that in Moloney MuLV (Mo-MuLV) ts3-infected cells, and in Rauscher MuLV (R-MuLV) ts17- and ts24-infected cells, increased amounts of intracellular viral Pr65 gag rapidly become associated with the cytoskeleton-enriched fraction during pulse labeling at nonpermissive temperature. Furthermore, examination of cell extracts from chase-incubated cells infected with these ts mutants revealed that Pr659 gag accumulated in the cytoskeleton-enriched fraction at 39° but not at 33°. During steady-state labeling, as much as half of the intracellular Pr65 gag becomes associated with the cytoskeleton-enriched fraction (i.e., is not solubilized by NP-40) at 39°. At permissive temperature only 10–15% of the intracellular Pr65 gag is cytoskeleton associated. In contrast, cells infected with R-MuLV ts25 or ts26 showed little or no preferential localization of Pr65 gag in the cytoskeleton-enriched cell fraction during a short pulse at 39°, but Pr65 gag accumulated in both the NP-40-soluble and -insoluble fractions during a chase incubation relative to the condition at 33°. Based upon these and previous results (Edbauer and Naso,1983), models for retrovirus assembly are described in which the association of Pr65 gag with the cell membrane and cytoskeleton plays a critical role in virus assembly, budding, and postbudding maturation.

Studies on the biosynthesis of cartilage proteoglycan in a model system of cultured chondrocytes from the Swarm rat chondrosarcoma.

Biosynthesis of cartilage proteoglycan was examined in a model system of cultured chondrocytes from a transplantable rat chondrosarcoma. Extensive modification with the addition of chondroitin sulfate glycosaminoglycan, N-linked oligosaccharide, and O-linked oligosaccharide is required to convert a newly synthesized core protein precursor into a proteoglycan. Kinetic analyses revealed the presence of a large pool of core protein precursor (t 1/2 approximately 90 min) awaiting completion into proteoglycan. The large t 1/2 of this pool allowed kinetic labeling experiments with a variety of radioactive precursors to distinguish between early biosynthetic events associated primarily with the rough endoplasmic reticulum from late events associated primarily with the Golgi apparatus. The results of a series of experiments indicated that the addition of N-linked oligosaccharide chains occurs early in the biosynthetic process in association with the rough endoplasmic reticulum, whereas the initiation and completion of O-linked oligosaccharides occurs much later, at about the same time as chondroitin sulfate synthesis. This also indicated that keratan sulfate chains, when present in the completed molecule, are added in the Golgi apparatus, as they are probably built on oligosaccharide primers closely related to the O-oligosaccharide chains. Furthermore, when 3H-glucose was used as the precursor, the entry of label into xylose, the linkage sugar between the core protein and the chondroitin sulfate chain, was found to occur within 5 min of the entry of label into galactose and galactosamine in the remainder of the chondroitin sulfate chain. This indicated that the initiation and completion of the chondroitin sulfate chain occurs late in the pathway probably entirely in the Golgi apparatus. Thus, proteoglycan synthesis can be described as occurring in two stages in this system, translation and N-glycosylation of a core protein precursor which has a long half-life in the rough endoplasmic reticulum, followed by extensive rapid modification in the Golgi complex in which the majority of glycosaminoglycan and oligosaccharide chains are added to the core protein precursor with subsequent rapid secretion into the extracellular matrix.

Biosynthesis of O-linked oligosaccharides on proteoglycans by chondrocytes from the swarm rat chondrosarcoma.

The core protein of proteoglycans from cartilage is substituted with glycosaminoglycans as well as N- and O-glycosidically linked oligosaccharides. We have taken advantage of the long intracellular half-life of the core protein precursor to the rat chondrosarcoma proteoglycan to study the temporal relationship between the addition of the chondroitin sulfate chains and the O-linked oligosaccharides onto the core protein during the formation of the completed proteoglycan molecule. Chondrocyte cultures were pulsed on day 2 with [6-3H]glucosamine for times ranging from 30-420 min. Media and corresponding 4% zwittergent, 4 M guanidine HCl extracts were then pooled and subjected to dissociative density gradient ultracentrifugation to yield purified proteoglycan monomers which were then subjected to alkaline borohydride treatment. The released chondroitin sulfate chains were then purified by precipitation with 50% (v/v) ethanol. The O-linked oligosaccharide-alditols in the supernatant fractions were purified by molecular sieve chromatography on Bio-Gel P-6, and analyzed after digestion with alpha-neuraminidase and subsequent chromatography on Bio-Gel P-2. The different O-linked oligosaccharide-alditols were identified from their hexosamine and hexosaminitol contents. The kinetics of entry of 3H label into N-acetylgalactosamine of chondroitin sulfate was indistinguishable from that into either N-acetylglucosamine or N-acetylgalactosaminitol residues of the oligosaccharide-alditols, with half-times to linear incorporation of 10-17 min. These results show that initiation as well as completion of the O-linked oligosaccharides on the core protein occurs essentially at the same time that chondroitin sulfate chains are added. The results suggest that these biosynthetic processes occur in the Golgi apparatus during the last few minutes of the total intracellular dwell time (half-time of about 90 min) of the core protein acceptor.

Comparative tryptic peptide analysis of candidate P85 gag-mos of ts1 10 moloney murine sarcoma virus and P38-P23 mos gene-related proteins of wild-type virus

The translation products of ts110-MuSV RNA were analyzed and compared to the candidate mos gene products of the wild-type virus, MuSV-349. ts110-MuSV-MuLV virion preparations contain two mos gene-containing RNA species, 4.0 and 3.5 kb in length, which are translated into the 85,000 molecular weight ( M r) candidate gag-mos protein (P85) and P58 gag and a third RNA, about 8.3 kb in length, representing the helper virus RNA. This latter RNA is translated into the core protein precursor Pr63 gag Translation of subgenomic poly(A)-containing ts110-MuSV RNAs produced polypeptides of 33,000 M r (P33) and 23,000 M r (P23), but yielded no P85 or P58 gag . ts110 P33 and P23 were found to be similar in both size and structure to P33 and P23 translated from 22S poly(A)-containing RNA from wild-type MuSV-349 RNA. One important difference between ts110 and MuSV-349 was the lack of a major 38,000 M r (P38) polypeptide in the ts110 translation products. It has been shown by others that the mos genetic region of MuSV-124 genomic RNA can be translated into a family of mos gene-derived polypeptides ranging in size from 43,000 to 18,000 daltons, and we have previously reported similar candidate mos gene products of 38,000 (38K) and 23K in the translation products of subgenomic poly(A)-containing RNA of MuSV-124 (Lyons et al., 1980). The same family of mos gene products is present in the translated product of MuSV-349 RNA. While highly related to one another, MuSV-349-coded P38 and P33 contained at least three additional tyrosine tryptic peptides not found in P23, whereas P23 contained at least one tryptic peptide not present in either P38 and P33. The tryptic peptide profiles of ts110-coded proteins, considered together with other results, indicated that P85 is a hybrid polyprotein containing p15, pp12, and part of p30 joined to a P33 MuSV-349 candidate mos gene product. In contrast, ts110 P58 gag is clearly a gag-gene product and shared no tryptic peptides in common with the MuSV-349 candidate mos gene products.

Effects of interferon on the expression of mouse mammary tumour virus in GR cells.

The effects of interferon treatment on the expression of murine mammary tumour virus (MuMTV) by a continuous mammary tumour cell line of GR mouse were investigated. Interferon markedly inhibited the excretion of MuMTV particles in the culture media as measured by hybridization of virus-specific RNA and virus-associated reverse transcriptase activity. There was no inhibition of virus RNA and protein synthesis in those conditions. The steady-state level of intracellular virus RNA and its rate of synthesis were not modified by interferon treatment. The intracellular levels of the major virus envelope and core proteins as measured by immunoprecipitation techniques remained unchanged. Interferon failed to inhibit the synthesis and the processing of the gp52 glycoprotein and p27 core protein precursors. However, the rate of maturation of the glycoprotein precursor was slowed down. Surface labelling of intact cells did not reveal accumulation of virus proteins on the cell membrane upon interferon treatment. The intracellular level of MuMTV-characteristic reverse transcriptase activity was reduced in interferon treated cells, although to a lower extent than extracellular virion-associated enzyme activity. MuMTV particles released from interferon-treated cells revealed no difference in their protein composition. These results are consistent with an inhibition by interferon of a late step in the replication of MuMTV.

Identification of core protein, an intermediate in proteoglycan biosynthesis in cultured chondrocytes from the Swarm rat chondrosarcoma.

After incubating cultured chondrocytes from the Swarm rat chondrosarcoma for 30 min with [3H]serine, a labeled macromolecule was found predominantly as Mr = approximately 370,000 species which was subsequently identified as a core protein precursor to cartilage proteoglycan from the following properties: (a) it was immunoprecipitated along with completed proteoglycan from cell extracts by an antiserum to the complex of hyaluronic acid-binding region, link protein, and hyaluronic acid. Its immunoprecipitation could be inhibited completely by the addition of purified hyaluronic acid-binding region to the extracts, indicating the presence of common antigenic determinants with this region of the proteoglycan core protein. (b) the core protein precursor was able to interact with the hyaluronic acid and link protein in proteoglycan aggregates added as carrier to extracts to form mixed aggregates of high buoyant density in associative CsCl density gradients. Labeled core protein precursor and link protein were subsequently isolated from the mixed aggregates from the top of dissociative CsCl density gradients. (c) radioactivity in core protein precursor after a 30-min pulse of [3H]serine disappeared after inhibiting further protein synthesis with cycloheximide concurrent with the appearance of label in completed proteoglycan molecules.